Cytokines, hormones and interactions with their cognate receptors play critical roles in the regulation and homeostasis of various physiological systems. Their dysregulation may lead to diseases related to metabolism, cancer development, and immune or hematopoiesis disorders.
Interactions between hormones, cytokines and their cognate receptors are complex and may require the implication of various co-receptors that modulate in a specific cell type their affinity and the signal transduction. Therefore, depending on the co-receptors involved, hormones/cytokines may compete with each other and may transduce either positive or negative signals for cell growth, survival and specific functions.
Leptin (Lep/OB) is a cytokine-like hormone mainly produced by adipocytes and plays a crucial role in the maintenance of energy balance through its effect in reducing food intake and increasing energy expenditure. It activates receptors (OBR) highly expressed in the brain hypothalamic arcuate nucleus (ARC), a site well known to control body weight (Heike Münzberg and Christopher D Morrison, Metabolism 2014). Hence, defect in leptin receptor signaling leads to severe obesity. Currently, two isoforms of the leptin receptors (OBR) are mainly studied, the short isoform OBRa and the long isoform OBRb.
Leptin mediates most of its effect by activating the long receptor isoform (OBRb), which is the dominant signaling species highly expressed in the brain hypothalamic arcuate nucleus (ARC) (Heike Münzberg and Christopher D Morrison, Metabolism 2014).
However, recent evidences showed that OBR is also expressed in peripheral tissues and that leptin is a more pleiotropic hormone playing a role in other pathophysiological processes including immune regulation, hematopoiesis, cancer development, and metabolism associated disorders including type 2 diabetes, kidney function failure and metabolism associated and age related ocular diseases.
Studies from our group, deciphering the role of adipocytes on the hematopoiesis regulation showed that neuropilin-1 (NRP-1) is overexpressed in femoral fatty bone marrow (Zakia Belaid et al. Heamatologica 2005). NRP-1 is a transmembrane glycoprotein implicated in axonal guidance and angiogenesis through specific ligands and co-receptors SEMA/Plexin and VEGF/VEGFR, respectively (Fujisawa H et al, J Neurobiol 2004 and Soker S et al, Cell 1998). NRP-1 has been shown by our group to be involved in 1) the immune response by participating in the formation of the immune synapse between dendritic cells and T-lymphocytes (R. Tordjman, Yves Lepelletier et al., Nature Immunology 2002), 2) the HTLV1 entry in lymphocytes (David Ghez, Yves Lepelletier et al., Journal of Virology 2005) and 3) the regulation of hematopoiesis by blocking granulopoiesis through the inhibition of the production of granulocyte colony stimulating factor (G-CSF) by macrophages under the influence of adipocytes (Zakia Belaid-Choucair et al. Stem Cell 2008). The latter function was independent of known NRP-1 ligands but involved leptin produced by adipocytes (unpublished data).
NRP-1 needs to form a complex with receptors belonging to the plexin family, which serve as the signal-transducing element for the axonal repulsion and collapse of neuronal growth cones after SEMA binding to the NRP-1/Plexin complex (He and Tessier-Lavigne, Cell 1997). NRP-1 may also interact with VEGF receptors (VEGF-R) forming a complex, which can be activated by VEGF165 for normal developmental angiogenesis (Soker S et al. Cell 1998). Based on both the literature and our results in granulopoiesis regulation, We postulated that the leptin receptor OBR may form a complex with NRP-1. Interestingly, macrophages involved in granulopoiesis inhibition expressed both NRP-1 and OBR at their cell surfaces. NRP-1 and OBR interaction in macrophages was detected by western blotting after NRP-1 co-immunoprecipitation.
By using a well described MDA-MB231 (NRP-1 positive and OBR positive) and T47D (OBR low and NRP-1 negative) breast cancer cell lines transduced either by shNRP-1 or cDNA encoding for NRP-1 we have shown that 1) NRP-1 forms a complex with OBR 2) NRP-1/OBR complex formation is leptin dependent 3) the NRP-1/OBR complex translocates to the nucleus 4) both complex formation and nuclear translocation are dependent on NRP-1 and JAK2 phosphorylation by the Serine/Threonine casein kinase2 (CK2). This finding was confirmed by the inhibition of CK2 by 3 different chemical compounds (TBB, DRB and CX4945) and by RNA silencing that prevented not only NRP-1 and JAK2 phosphorylation but also the formation of the NRP-1/OBR complex.
Leptin has been reported to regulate more than 64 genes including those for growth, cell cycle regulators, extracellular matrix proteins and gene associated with metastasis (Perera C. N et al. J of Endocrinol 2008 and EBM 2008).
Besides a major role in energy homeostasis, the adipokine hormone-like “Leptin” is emerging as a pleiotropic cytokine with pro-angiogenic activity by inducing VEGF and VEGFR2 expression. Multiple signaling molecules modulate the complex interplay between the vascular system and the adipocytes (Yihai Cao, Cell Metabolism 2013).
In breast cancer, leptin is considered as a pro-angiogenic marker (Ruben Rene Gonzalez-Perez et al, Cancers 2013). Anti-VEGF (bevacizumab) treatment of patients with rectal cancer was significantly associated with distant metastasis at three years, which correlated with the up-regulation of NRP-1 and activation of inflammatory pathways (Xu Lei et al, cancer Research 2009).
Interestingly, our functional characterization of NRP-1/OBR complex using breast cancer xenograft model led us to demonstrate that NRP-1 modulates leptin function by decreasing cell proliferation, inducing cell migration and lymph node infiltration. Since leptin has been reported to be pro-angiogenic, we postulated that physiologically, VEGF (a target of Bevacizumab) might play a negative feedback on leptin action during angiogenesis. During treatment with Bevacizumab, VEGF might be unable to reduce leptin action and thus NRP-1 overexpression will be in the favor of NRP-1/OBR and leptin interactions. This complex would lead to a non-canonical signaling involved in metastasis and thus decrease of the overall survival.
As hypothesized, preventing the binding of VEGF to VEGFR by treatment of MDA-MB231 breast cancer cell line with Avastin alone induces in vitro cell migration, as did leptin alone. This effect was further increased when the cells were co-treated with both Avastin and leptin. The increase of leptin effect on cell migration by Avastin treatment raised the question whether VEGF may have a negative feed-back on NRP-1/OBR signaling. Interestingly, we could demonstrate that VEGFs bind to OBR. Thus, we can conclude that blocking VEGF with Avastin increases leptin action and induces metastasis and other known effect of leptin such as inflammation that is also reported during Avastin therapy through an increase of NRP-1OBR complex formation. Interestingly our hypothesis is clearly demonstrated by the increase of NRP-1/OBR complex detection by PLA-HRP technology in MDA-MB231 breast cancer cell line treated with Avastin compared to the control cell. The experiment was done in the presence of normal human serum in order to mimic the physiologic condition during Avastin therapy.
By using a BioLayer Interferometry technology (BLI, http://www.fortebio.com), we were able to demonstrate a direct interaction between recombinant proteins Leptin and NRP-1. In contrast to other NRP-1 ligands such as VEGF, and Sema3A known as competitors, leptin binds directly to NRP-1 but do not compete with VEGF binding. Similarly, VEGF does not prevent leptin binding to NRP-1. These observations suggest that leptin and VEGF have distinct binding domains.
This finding suggests that anti-VEGF therapy should be combined with an anti-leptin therapy in cancer.
This could also implicate disease associated to immune system dysfunction where leptin has been clearly associated in the disease aggravation.